Alternative pre-mRNA splicing is used widely among eucaryotes in the control of both gene expression and function. Regulation of splicing plays a critical role in a wide variety of processes needed for normal development. Despite its importance in gene regulation, remarkably little is known about the molecular mechanisms that result in alternative splicing. Particularly lacking is information on the biochemical activities of the trans-acting regulators that are thought to direct usage of alternative splicing pathways. Very few such regulators have yet been identified and, of those known, only a few have been studied in sufficient detail to yield significant information about their mechanisms of action. We propose to investigate the molecular mechanism by which transformer-2 (tra-2), an established splicing regulator from Drosophila melanogaster, functions to repress the splicing of an intron found within the tra-2 pre- mRNA itself. We will first investigate the physical interaction of tra-2 protein and pre-mRNA by using a UV crosslinking assay to identify both RNA and protein sequences needed for binding. The significance of the identified binding sites with respect to regulated splicing will be evaluated in the developmental context of the fly's male germline where autoregulation normally occurs. This will be accomplished in experiments using stable transgenic fly strains in which the binding site sequences have been altered. Additional studies will follow up on our preliminary finding that indicate that a general factor plays an important role in this binding interaction. We describe an approach for both identifying this factor and assessing its role in regulation of splicing. Finally, we will use a cell free in vitro splicing system to examine how tra-2 protein molecules interfere with the splicing of the M1 intron. The aim of these studies will be to identify the specific interactions between the splicing machinery and the pre-mRNA that are blocked in the presence of RNA bound tra-2 protein molecules. The results of these studies will provide substantial insights into the mechanism by which tra-2 protein molecules affect splicing. Such insights should prove helpful in understanding how splicing regulators function to control alternative splicing in other systems.